Among biopharmaceuticals, therapeutic agents using antibody are being most actively studied, which bind specifically to targets (i.e., antigens) that are expressed specifically in certain diseases. Particularly, identification of tumor-related antigens that are expressed on the surface of cancer cells is being actively performed, and methods of diagnosing and treating tumors using antibodies (i.e., anticancer antibodies) that bind to the antigens to inhibit cell growth or induce cell death are being widely used, and the field of these methods also has a very good prospect.
Such anticancer antibodies have very high target specificity, but their cytotoxic effects on the cancer cells are usually lower than those of existing cytotoxic drugs (i.e., anticancer drugs or agents). So in many cases, these anticancer antibodies are subject to a combination therapy with cytotoxic drugs or other cell proliferation inhibitory drugs.
In connection with the combination therapy as described above, there have been active studies on a modified antibody-drug conjugate (mADC), in which the therapeutic effect of the bound-cytotoxic drugs increases while the toxicity decreases. It is recognized that when the modified antibody-drug conjugate is used, the systemic toxicity of the drug can be reduced and the cytotoxicity of the drug can be enhanced specifically in cells (particularly cancer cells) having a target overexpressed therein, thereby increasing the therapeutic effect of the drug.
Indeed, modified antibody-drug conjugates comprising a cytotoxic drug or radioisotope conjugated to an antibody, such as ZEVALIN™ [Witzig et al., J. Clin. Oncol, 2002, 20(15): 3262-3269]) or MYLOTARG™ [Drugs of the Future, 2000, 25(7):686]), have been successfully developed for the treatment of non-Hodgkin lymphoma or acute myeloid leukemia. In addition, many attempts have been made to conjugate highly toxic mertansine (such as cantuzumab mertansine (Immunogen, Inc. [Xie et al., J. of Pharm. and Exp. Ther. 2004, 308 (3):1073-1082]) or trastuzumab mertansine (Roche [Isakoff et al., J. Clin. Oncol. 2011, 29(4): 351-4])) to an antibody or conjugate other cytotoxic drugs, for example, dolastatin derivatives such as auristatin peptides, auristatin E (AE), monomethyl auristatin (MMAE) or MMAF to antibodies like cBR96 (specific to Lewis Y on carcinomas), cAC1O which is specific to CD30 on hematological malignancies, or anti-CD20 antibody for treatment of CD20-expressing cancer, or Rituxan for immune disorders, anti-EphB2R antibody for treatment of colorectal cancer, 2H9, anti-IL-8, or E-selectin antibody ([Klussman, et al., Bioconjugate Chemistry, 2004, 15(4):765-773]; [Doronina et al., Nature Biotechnology, 2003, 21(7):778-784]; [Francisco et al., Blood, 2003, 102(4):1458-1465]) US 2004/0018194 A1) WO 04/032828 A3; [Mao et al., Cancer Research, 2004, 64(3):781-788]; [Bhaskar et al., Cancer Res, 2003, 63:6387-6394]).
In addition, an attempt has also been made to develop modified antibody-drug conjugates using daunomycin, doxorubicin, methotrexate or vindesine. It is known that bacterial toxins such as diphtheria toxin, plant toxins such as ricin, or small molecules such as geldanamycin ([Mandler et al., J. of the Nat. Cancer Inst, 2000, 92 (19):1573-1581]; [Mandler et al., Bioorganic & Med. Chem. Letters, 2000, 10: 1025-1028]; [Mandler et al., Bioconjugate Chem, 2002, 13:786-791]), maytansinoid ([EP 1391213 A1]; [Liu et al., Proc. Natl. Acad. Sci. USA, 1996, 93: 8618-8623]) or calicheamicin ([Lode et al., Cancer Res, 1998, 58: 2928]; [Hinman et al., Cancer Res, 1993, 53: 3336-3342]) may be used as drugs in antibody-drug conjugates. These cytotoxic drugs exhibit cytotoxic and cell proliferation inhibitory effects by mechanisms such as tubulin binding, DNA binding or topoisomerase inhibition.
When a conventional process that was used to induce covalent linkage between a drug and an antibody is employed to prepare the antibody-drug conjugate as described above, the drug will be bound to a number of sites in the antibody, producing a heterogeneous mixture. For example, a cytotoxic drug is likely to be bound to an antibody through a number of lysine residues contained in the antibody to produce a heterogeneous antibody-drug conjugate mixture. Also, the heterogeneous mixture might have different drug binding distribution ranging from 0 to about 8 depending on reaction conditions, which means that the number of drug molecules bound per unit antibody varies.
Also for the antibody drug conjugate having the defined number of drug binding, there might be another potential heterogeneity, depending on the various conjugation sites. Homogeneous purification from this heterogeneous mixture is not suitable for use in the mass production of medicines ([Hamblett et al., Clin. Cancer Res, 2003, 10, 7063-7070], [Wang et al., Protein Sci. 2005, 14, 2436-2446]).
Another method for conjugating a drug to an antibody is reducing disulfide bonds between cysteine residues in antibody by reducing agents, and then conjugating drugs to free thiol groups of the reduced cysteine residues. This method also has disadvantages in that the inherent characteristics of the antibody can be lost and a heterogeneous mixture is produced in large amounts. Specifically, immunoglobulin M is an example of a disulfide-linked pentamer, while immunoglobulin G is an example of a protein with internal disulfide bridges bonding the subunits together. In such proteins, reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) or selenol generates reactive free thiols ([Singh et al., Anal. Biochem. 2002, 304:147-156]). This approach may result in loss of antibody tertiary structure and antigen binding specificity ([Jagath et al., Nature Biotechnology, 2008, 26(8):925-32]).
The representative disadvantage of the conventional antibody-drug conjugation method as described above is that it is difficult to precisely control the drug conjugation sites in antibody and the number of conjugated drugs. In an attempt to overcome this problem and to introduce a free thiol group, a specific amino acid was substituted with cysteine where the substituted cysteine did not impair the function of an antibody. For this purpose, a screening method for optimum cysteine mutant was developed after predicting the reactivity of thiol group in each possible mutating site in antibody (Korean Patent Laid-Open Publication No. 2007-0054682, ‘ThioFab technology’). An antibody-drug conjugate of trastuzumab mertansine produced by this method is in clinical trials for treatment of metastatic breast cancer ([Burris III et al., J. Clin. Oncol, 2011, 29(4):398-405]). The above-described ThioFab technology has an advantage in that damage to a disulfide bond in a parent antibody can be minimized by introducing a new cysteine into the antibody, but the concern about modification of the structure and the function of the parent antibody still remains, because some amino acids in the parent antibody are mutated by cysteine.
Accordingly, there is an urgent need for the development of a novel antibody-drug conjugate and a production method thereof, in which the number and position of drug molecules conjugated to a parent antibody can be accurately controlled while retaining the structural and functional characteristics of the parent antibody.